CHARACTERISATION AND MOLECULAR TYPING OF CLINICAL AND ENVIRONMENTAL ISOLATES OF VIBRIO PARAHAEMOLYTICUS

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Vibrio parahaemolyticus is a natural inhabitant of coastal waters worldwide and is the
leading cause of seafood-borne gastroenteritis. This study reports on the use of several
molecular characterisation methods to screen clinical and environmental isolates of V
parahaemolyticus to assess whether such techniques can be used to distinguish pathogenic
isolates reliably.
In a total of 86 isolates mainly of V parahaemolyticus but also including V cholerae, V
vulnificus and several other species, serotypes of the more virulent clonal group 03:K6
were identified, but otherwise there appeared no association with serotype and phenotype.
The tdh and trh genes encoding haemolysins that are typically associated with virulent
isolates were found in a significantly large number of isolates; however, poor concordance
between haemolytic activity and the presence of the gene tdh was found. In an effort to
establish more accurate relationships amongst clinical and environmental isolates of V
parahaemolyticus, four molecular typing systems were employed; namely pulsed-field gel
electrophoresis (PFGE), intergenic transcribed spacer (ITS) analysis, tDNA intergenic
length polymorphisms (tDNA-ILPs) and randomly amplified polymorphic DNA (RAPD).
Typing patterns and clustering analysis using these methods differentiated V
parahaemolyticus from other marine species as well as at the subspecies level. PFGE with
NotI was shown to be the most discriminative but suffered from not being universally
applicable. Both ITS and tDNA-ILP methods were sufficiently discriminatory with
discrimination indices (DI) of between 0.568 and 0.724, depending on the primers
employed. The discriminatory ability of RAPD was also affected by the primers used (DI=
0.959 - 0.965) but closely matched that of PFGE (DI = 0.976). Additionally, both RAPD
methods were able to distinguish putative markers for the pandemic clonal group. Typing
systems appeared largely stable in duplicate and triplicate analyses with multiple primer
pairs with some obvious variability in the reproduction of faint amplicons. All methods
except PFGE were simple to execute but none of the methods could distinguish V
parahaemolyticus into obvious lineages based on the clinical or environmental source.
With the recent implication of a type Ill secretion system {TTSS) involved in the
pathogenicity of V parahaemolyticus, a multiplex PCR system using PCR primers that
spanned both TTSSl and TTSS2 regions was developed. Dot-blot analysis confirmed
TTSS2 genes in at least 30% of environmental isolates. Nucleotide sequence analysis
revealed l00% sequence homology in three loci of TTSS2 putative structural genes. In
comparison, a total of 34 single nucleotide polymorphisms (SNP) were identified in three
TTSS1 regions. In two of the regions, the SNPs were synonymous, whereas a non-synonymous
substitution in the structural gene vcrDI resulted in valine replacement with
isoleucine. In addition, nucleotide deletions in TTSS1 with resultant frameshift mutations
were identified. The finding that significant numbers of environmental isolates also possess
TTSS2 genes is contrary to currently held opinion that TTSS2 is only present in clinical
isolates. It is hypothesed that the high incidences of V parahaemolyticus infections may be
related to active TTSS2 genes, whereas a high degree of polymorphisms in TTSS1 suggest
it may be inactive.

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All items in PEARL are protected by copyright law.
Author manuscripts deposited to comply with open access mandates are made available in accordance with
publisher policies. Please cite only the published version using the details provided on the item record or document.
In the absence of an open licence (e.g. Creative Commons), permissions for further reuse of content should be
sought from the publisher or author.